DESCRIPTION The long-term goal of the original proposal, now in its second year, is to understand the mechanisms involved in protein deacylation by isolating and characterizing the enzyme(s) responsible for the turnover of palmitate covalently bound to a model palmitoylated protein, H-Ras. In the past two years, we have purified and molecularly cloned palmitoyl-protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and other proteins in vitro. From these experiments, we learned that PPT is secreted from cells and is unlikely to play a role in Ras plamitate turnover. However, we have recently demonstrated that mutations in the PPT gene cause the recessively-inherited neuronal degenerative disease, infantile neuronal ceroid lipofuscinosis (INCL). This disease is characterized by the accumulation of proteolipid material in storage bodies within cells, suggesting that PPT plays a crucial role in the catabolism of lipid-modified proteins. In the next funding period, we will continue to focus upon the molecular and cell biology of this novel enzyme. We propose to characterize the PPT gene, to explore protein structure-function relationships through the study of naturally-occurring and site-directed mutations, and to determine how the enzyme trafficks within the cell. We will also use cells derived from normal and INCL patients in metabolic labeling experiments to test the hypothesis that PPT plays a role in the catabolism of lipid-modified proteins. Finally, we will analyze the products of the enzymatic reaction of PPT and INCL cells or cell extracts to identify the most significant physiological substrates for PPT. We believe that this work may lead to new insights into cellular metabolism and the pathogenesis of neurodegenerative disease.